Abstract
BackgroundPyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation is generally considered as a safe, efficient, and inexpensive way to eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production.ResultsIn this study, random mutagenesis was performed on one pyrethroid-hydrolyzing esterase, Sys410, to enhance its activity and thermostability. Two beneficial mutations, A171V and D256N, were obtained by random mutagenesis and gave rise to the mutant M2. The mutant displayed ~1.5-fold improvement in the kcat/Km value and 2.46-fold higher catalytic activity. The optimal temperature was 10 °C higher than that of the wild-type enzyme (55 °C). The half-life at 40–65 °C was 3.3–310 times longer. It was surprising that M2 has a half-life of 12 h at 70 °C while Sys410 was completely inactivated at 70 °C. In addition, the desired gene was extracellularly expressed in a Pichia pastoris host system. The soluble expression level reached up to 689.7 mg/L. Remarkably, the enzyme could efficiently degrade various pyrethroids at moderate temperature for 15 min, exceeding a hydrolysis rate of 98%, which is the highest value ever reported.ConclusionsThis is the first report about random mutagenesis and secretory expression of pyrethroid-hydrolyzing esterase with high-level productivity and purity in P. pastoris. Broad substrate specificity, enhanced activity and thermostability make M2 an ideal candidate for the biodegradation of pyrethroid residues.
Highlights
No one enzyme has all the properties, such as high hydrolytic activity [22, 23], good stability [16, 23], and high productivity [17,18,19,20,21], which are indispensable for practical biodegradation
The results demonstrated that the ability of the enzyme from P. pastoris to degrade various pyrethroids was in good agreement with that expressed in E. coli BL21 (DE3)
Pyrethroids are potentially harmful to human health and ecosystems
Summary
It is necessary to develop some efficient strategies to degrade pyrethroid residues. Several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production. Recombinant enzymes have greater potential in eliminating pyrethroid residuals, especially with mass production and safety [16]. Almost all of them are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy deactivation and low production [24]. To facilitate downstream processing in large-scale biotechnological applications, secretion of the overexpressed enzymes into the culture medium is desirable [25, 26]
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